Antibiotic funginon and a process for producing, using aspergillus clav atus



Unitecl States Patent 3361629 ANTIBIOTIC FUNGINON ANI) A PROCESS FOR PRODUCING, USING ASPERGILLUS CLAVATUS Jiro Sawada, Kodaira, Sadafumi Omura, Tokyo, Hiroshi Nakayoshi, Chiba, Kazuo lrumura, Tokyo, and Toshiya Kitahara, Shinjuku, Tokyo, Japan, assignors to Taisho Pharmaceutical Co., Ltd., Tokyo, .aran a corporation of Japan Filed Aug. 7, 1964, Sar. No. 333,204 Claims prority, appiication Japan, Aug. 9, 1963, BIS/40,813 5 Claims. (Cl. 167-65) ABSI'RACT OF THE DISCLOSURE Funginon, havng a calculated empirical formula of C H O and a molecular weight of about 144 as meas ured cryoscopically, is a useful antibiotic. The ultraviolet absorption spectrum and infrared spectrum of the new antibiotic are shown respectively in FIG. 1 and FIG. 2 of the drawings.

This invention relates to a new antibiotic substance, and to a process for the production thereof.

More particularly, the invention re1ates to a new antibiotic substance designated funginon, possessing antimicrobial activities and therapeutically useful properties.

There has heretofore been little or no demonstration of antbiotic substance coupled with W toxicity and with antifungal, antibacteriai and anti-yeast activities or animals.

The object of the present invention is to obtain the most superior product comprising the above antibiotic activities. This has been accomplished by the isolation of a new microorganisrn which has pr0cluced a new su perior antibiotic substance as hereinafter described.

It is still another object of the invention to obtain easily the new and useful antibiotic substance designated fungnon, possessing superior activities coupled with low toxicity and potential value.

Funginon possesses several therapeutic properties, and is a medicament useful for therapy of etiological fungi disease, tuberculosis, etc.

The above-rnentioned new antibiotic substance, funginon, can be produced by cultivation of a new strain which resernbles Aspergz'llus clavatus or Aspergllus janus.

The said microor anism strain was isolated from a soil sample collected in Tokyo, Japan.

The microbiological properties of the new strain, Aspergillus clavatus TPR-32OZ (ATCC No. 15550) discovered and designated by the applicants are as follows:

MORPHOLOGIES General morphological observation was carried out upon the new strain culivated in Czapek-agar culture medium at 28 C. for 3 to 7 days.

Mycelium: 8 to 12;u in breadth, sepia, irregular branches,

flocculents.

Condiophore: usually enlargecl up toward the apex.

Foot-cell: mostly 500 by 5LL in small, 2500 by 2u in large, wal] smooth and frail.

Conidial heads clavate: 200 to 300 by 80 to 18u. in fresh culture for 3 to 5 days.

Colonies: White at first and greenish-blue in full growth, constituting conidiophore, small conidial heads globose and conidial heacls clavate hearing 50 to 7,LL

Vesicle cylirrdrical clavate: 180 to 200 by 21 to 63,.

Sterigmata clavate: 4 to 6.5,. in single series (however, when the Vesicle is exceptionally globose, sterigrnata Patented Jan. 2, 1968 Conidal globose: good growth upon maltose-agar culture.

Conidial clavate: goed growth upon the other culture.

As Inentioned above, Aspergllus clavatus TPR-3202 is characterized by two kinds of dilerent conidial heads and vesicles DEVELOPMENT IN CZAPEK-AGAR CULTURE MEDIUM The second days growth upon inoculating and cultivating Aspergllus clavalus TPR-3ZO2 at 25 C. showed shaped flocculated hyphae and the third days growth constituted Ioose conidial heads grayish-green or white, which were of smcoth surface and white in the surroundings and occasionally irregular. The seventh days growth of its cultivaton gave large colonies of 10 mm. in height and colorless back surface of colonies possessed insoluble pigment.

KOII EXTRACT-AGAR CULTURE MEDIUM Growth of spergillus clavatus TPR-3202 was seen with the naked eye upon cultivating at 25 C. for 2 days and it formed white velvet-like knolls and thereafter white cottonlike colonies.

The fourth days growth gave a constitution of greenishb1ue spores and greenish-blue all round colonies. The back surface of colonies showed light brown concentric circle figures. The tenth days growth gave grayish-white colonies covered by cotton-like hyphae.

RAULIN THOM CULTURE MEDIUM Upon cultivating at 25 C., the second days growth of Aspergillus clavalus TPR3202 gave thinly floeculated white colonies, and the fourth days growth constituted spore on the colonies colored to greenish-blue thick flocculent. The back surface of colonies was colored grayish brown and thereafter colonies constituted concentric circle growth.

IIALT EXTRACT-AGAR CULTURE MEDIUM Upon cultivating at 30 C. the second days growth gave white cotton-like colonies, and the third days growth constituted spores on the colonies colored greenish-blue. Further cultivation showed fiat colonies covered by short velvet-like aerial hyphae. In this state, long condiophores which possess greenish-blue conidial heads globose constituted sporadically on the colonies.

As a result of comparison of these distinguished characteristics of Aspergllus clavatus TPR-3202 by means of the classification described in Thorn and Rapers Manual of the Aspergilli (1945), the new strain is shown to be similar to the Aspergllus clavatus group. However, detailed comparison shows chat one of the characteristics of the newly discovered strain is based on constitut-ng small globose upon the clavate conidial heads. These conidial heads bear greenish-blue condia which are globose or sub-globose and srnooth wall, and occasionally possess two series sterigmata.

For example, the dstinct characteristics of the strain, Aspergillus clavatus TPR-3202, are shown in the followng:

Conidia: greenish-blue, surface smooth. Vesicle: clavate. Conidiophore: smooth.

Recapitulating, the said microorganism useful for the preparaton of fuginon has been designated Aspergllus clavatus TPR-3202, a newly-discovered strain similar to Aspergillus janus belonging to Aspergllus verscolor group, sydowz' series, according to Thorn and Rapers Cl'assification.

The newly-discovered strain, Aspergillus clavatus TPR-3202, can, however, be difierentiated from the strain of Aspergllus janus from the viewpoint of characteristics: conidial wall-smooth, larger conidial heads clavate comprising conidial heads globose, single series of the sterigmata hearing on the conidial heads clavate.

According to these observations, the strain of this i11- vention, Aspergllus clavaus TPR-3202, may also be considered to belong to the Aspergillus clavatus group, but t is distinguished from the authentic strain of the latter described in the literature in several characteristics. It may nevertheless be designated Aspergillus sp. TPR- 3202.

In accordance with one aspect of the present invention, the new antibiotic substance, funginon, can be protluced by cultivation of the microorganism, Aspergilus clavatus TPR-3202, in an appropriate nutrient medium under the same conditions as optimum cultivation of mold. The antibitic is similarly produced when using a mutant or variant of Aspergillus clavatus TPR-3202 produced from the latter by various means, such as X-ray ultra-violet radiation, etc.

Essentially, the medium contains a carbon source, a nitrogen sou'rce and trace inorganic elements.

Examples of suitable carbon sources are glucose, sucrose, lactose, maltose, soluble starch, dextrin, glycerol, etc. Suitable sources of nitrogen for the. process include ammonium salt, nitrate, corn steep liquor, meat extracts, yeast extracts, soybean meal, wheat gluten, cotton seed fiour, peptone, peanut meal, etc. Inorgarric salts as nitrogen sources in the nutrient medium give goed yield results for funginon.

Examples of suitable sources of trace inorganic elements are mineral salts, such as sodium chloride, calcium carbonate, potassium phosphate, zinc sulfate, ferrous sulfate, magnesium phosphate, etc. Presence of trace metals tends to enhance yields.

The cultivation of the newly discovered strain, Aspergillus clavaus TPR-3202, in the nutrient medium can be carired out in a number of difierent ways. For example, the microorganisrn can be cultivated under aerobic conditions on the surface of the medium (sulface culture) or it can be cultivated beneath the surface of the medium (deep culture), that is, in submerged condition, if oxygen is simultaneously supplied.

Themaximum yield of the antibiotic substance, funginon, can be obtained from the culture liquid wthin 8 to 13 days in the former mode of operation and within 6 to 8 days in the latter procedure, under optimum conditions at temperatures between about 25 to 30 C.

In order to isolate the eflective substance present in the culture mixture after completion of the fermentation phase of the process, the conventional manner usually used for the isolation of effective components contained in Various fermentation mixtures may be conveniently employed.

O11 isolatng funginon from the above-mentionecl sources, such expedients as concentration of the efiective substance, removal of impurities and conversion to a composition capable of being more readily purified are used, in adaptation to the properties of funginon. 'hus, use may be made, for examples of absorption agents in water.

As shown in the accompanying FIG. 1, the maximum ultraviolet absorption shows E =723 at 273-263 I.I1p

in methanol.

Funginon is soluble in water, methanol, ethanol, acetone, ethylacetate, butylacetate, glacial acetic acid, ether, pyridne and dioxane, slightly soluble in chloroform, benzene and insoluble in carbon tetrachloride, petroleum ether and hexane.

Thus-obtained funginon is stable in the acidic or neutral aqueous solution in pH range of 2.0 to 7.0 at C. There has been shown no decreasing of its antibiotic activity upon heating for 30 minutes. As result of cryoscopic method, the molecular weight of funginon is 144 by using dioxane.

Elemental analysis gives the following results: (1) C, 54.77; H, 5.44; 0, 39.79 (by dierence) (first). Analytical value: (2) C, 53.34; H, 5.33; 0, 41.31 (by difierence) (second).

The empirical formula C H O corresponds to the analytical and molecular weight data, supra.

It gves positive nitro-prusside, Molisch, Benedict, Nylander, diazo, Fehling and Nessler tests and negative ferric chloride and buret tests, and produces a large amount of brownish-black color precipitaton while making no silver mirror by Tollens test.

LD tests in mice of funginon show the acute toxicty to be about 20.2 mg./kg. of body weight when given intraperitoneally, 24.6 mg./kg when given intraveneously, and 108 mg./kg. when administered orally.

Funginon shows activities against various microorganisms and the following table illustrates the antibiotic spectra of funginon:

ANTIBIOTIC SPECTRA OF FUNGDNON Min. inhibitory Test microorganisms: c0n. (mcg./ ml.)

1. Sarcna lutea 12.5 2. Bacllus subtils 50 3. Bacillus megatherium 3 4. Mz'crococcus flavus 100 5. Serratg marcescens 50 6. Prteus vulgaris HX-l9 25 7. Klebsiella pneum0nae 50 8. Psudomomzs aerugnosa 100 9. Shigella flexineri KB- 12.5 10. Slzgella flerineri KBIII 12.5 11. Salmonella paratyphi A 25 12. Salmonella purafyphz B 12.5 13. Salmonella typhi H-901 12.5 14. Escherchia c0l O55 12.5 15. Streptococcus py0genes 50 16. Dplococcus pnumaniae 50 17. Aspergillus ryzae IAM2683 100 18. Aspergllus m'ger 100 19. Pencllium chrysogenum 100 20. Trchphyt0n ntergzale 6 21. T richophyton rubrum 50 22. Trch phyton aster0ides 12.5 23. Mcrosporum cam's 25 24, Sporozrchum schenki 25 Min. inhibtory From the above-mentioned results, it is seen that funginon is a relatively W toxic and useful medicament possessing high activity against etiological fungi and tuberculosis germs.

Growth-inhibiting tests against Ehrlichs ascites tumor cells were carried out as ollows:

Used animals were selected from 4 to 5 week-01d mice weighing about to g. 200 million of mouse-Ehrlichs ascites tumor cells were given intraperitoneally to the above described rnice respectively.

After 24 hours frorn infecting with tumor, intraperitoneal administration of 4 mg./kg./day (80 mcg./mouse/ day) and 8 rng./kg./day (160 mcg./mouse/day) of funginon was continued for a week to the infected mice. As results of the tests following 40 days observation period, funginon is shown to be etective by the intraperitoneal route at a dose of 8 mg./kg./day at which 90% maintenance is obtained for the infected animal groups with tumor and 50 to 60% maintenance is obtained with intraperitoneal administration of 4 mg./kg./day. The mice thus treated with funginon completely resist increase of infection with ascites tumor.

In view of the oregoing result, funginon is a novel antibiotic substance and is chemically and biologically dilerent from known antibiotics, antimolds and other substances. It is of good eiect in therapeutic use.

The antibiotic of the instant invention is also useful in separating and/or classifying mixtures of microorganisms for biological research and medical diagnostic purposes. It is further useful as a disinfectant agent for protecting against contamination of e.g hospital rooms or apparatus by pathogenic microorganisms such as Mycobacerz'um tubercul0sis H37V and Trchop/zytan interdigzale. It can also be employed in protectng various substrates, such as fabn'cs, against fungus attack.

The following example illustrates a suitable method for preparing, purifying and fractionating of funginon, but is not to be construed as a limitation of this invention, variation being possible within the spirit and scope of this invention.

Extlmple 1 An aqueous nutrient medium (45 liters) is prepared with the following ingredients:

Percent by weight The nutnent medium is placed and sterilized in a jar ermentor. Then the nutrient medium is inoculated with the funginon-producing strain, Aspergillus clavatus TPR- 3202, and cultivated under 45 liters/min. of sterilized aeration at 28 C., with stirring at 300 r.p.m.

The cultivation is carried out for 7 days until the antibiotic substance, funginon, attains maximum yield. After completion of the fermentation, 2% by weight of diatomaceous earth is added to the fermentation broth containing funginon and the mixture is filtered with suction in order to isolate the eiective substance.

On the other hand, into the filtrate is added 2% by weight of actve-carbon.

The active-carbon absorbed funginon is collected and extracted with 80% acetone. After distilling ott the acetone under reduced pressure, the aqueous solution is subjected to extraction with five-old volume of ethylacetate.

A residual syrup, obtained by distilling off ethylacetate under reduced pressure, is further extracted with ether, and Na SO is added to dehydrate the ether solution. The dehydrated matter is then poured onto a column containing chromatographic active alumina. Funginon is spotted at the top of the column.

The spotted column is then washed with ether, followed by developing with aqueous saturated ethylacetate. The first active ethylacetate fractions colored light yellow are collected and subjected to distilling ofi ethylacetate under reduced pressure. Fuginon is obtained as yellow oily matter weighing about 7.7 grams (yield The significant maxima of the IR spectrum are as follows: IR maxima wave numbers: 3380 cm. 1680 cm. ,1250 cm. 1035 cm. 2930 cm. 1450 cm. 1220 cm. 1005 cm. 1775 cm. 1403 cn1. 1173 cm. 877 cm. 1750 cm. 1375 cm. 1075 cm. 829 cm. 785 cm. cf. FIG. 2 of the drawings.

What is claimed is:

1. An antibiotic substance, funginon, eective in inhibiting the growth of Gram-positive and Gram-negative microorganisms and Ehrlichs ascites tumor cells, said antibiotic substance being a yellow oily matter, and being soluble in water, methanol, ethanol, acetone, ethylacetate, butylacetate, glacial acetic acid, ether, pyridine and dioxane, slightly soluble in chloroform and in benzene and almost insoluble in carbon tetrachloride, petroleum ether and hexane; gving a brownish black color but forrning no slver mirror by Tollens test, giving positive nitro-presside, Molisch, Benedict, Nylander, diazo, Fehling and Nessler tests and giving negative ferric chloride and Biuret tests; havng a degree of optical rotation [oc] =+45.55 (in water); contaning the elements carbon, hydrogen and oxygen, having analytical value:

1 c, 5477; H, 5.44; 0, 39.79 (by dierence), (2) C, 53.34; H, 5.33; 0, 41.31 (by ditference),

having a molecular weight of 144 and corresponding to the empirical ormula C H 0 and showing the ultraviolet absorption spectrum and the infrared absorption spectrum as in the attached drawings, FIG. 1 and FIG. 2 respectively.

2. A process for the production of funginon which comprises cultivating the microorgansm strain Aspergllus clavatus TPR-3202 (ATCC No. 15550) under aerobic conditions at a temperature of about 28 C., in an aqueous nutrient medium containing an assimilable carbon source, a nitrogen source and inorganic elements, recovering the unginon-containing mixture so-produced from the medium, and separating funginon from the said mixture.

3. The process of claim 2, wherein the carbon source is selected from the group consisting of glucose, sucrose, lactose, maltose, soluble starch, dextrin and glycerol.

4. The process of claim 2, wherein the nitrogen source is selected from the group consisting of ammonium-salt, nitrate, corn steep liquor, meat extracts, yeast extracts, soybean meal, wheat gluten, cotton seed fiour, peptone and peanut meal.

5. The process of claim 2, wherein the trace inorganic element is selected from the group consisting of sodium chloride, calcium carbonate, potassium phosphate, zinc sulfate, ferrous sulfate and magnesium phosphate.

References Cited UNITED STATES PATENTS 9/1963 Olson 16765 9/1963 Olson et al. 80 

